鸭坦布苏病毒诱导的宿主天然免疫反应及与RLR信号通路的互作研究

发布时间：2018-04-10 04:30

本文选题：鸭坦布苏病毒　切入点：天然免疫反应　出处：《山东农业大学》2017年博士论文

【摘要】：鸭坦布苏病毒(duck Tembusu virus,DTMUV)是一种有囊膜的,单股正链RNA病毒,属于黄病毒科黄病毒属。DTMUV于2010年首先在福建、浙江等地的发病鸭场中分离到,主要引起产蛋鸭群产蛋下降、商品肉鸭生长迟缓和神经症状。该病传播迅速,给我国养鸭业造成了巨大的经济损失。宿主天然免疫反应在病原微生物感染早期发挥重要作用,模式识别受体(pattern recognition receptors,PRRs)作为宿主天然免疫系统的关键组成元件,主要识别病原微生物的保守成分。RIG-I样受体(RIG-I like receptor,RLR)是主要分布于非免疫细胞中的一类胞浆PRRs,包含维甲酸诱导基因I(retinoic acid induced gene-I,RIG-I)、黑色素瘤分化相关基因(melanoma differentiation associated gene 5,MDA5)以及遗传和生理学实验基因2(laboratory of genetics and physiology-2,LGP2),主要识别RNA病毒,通过接头分子线粒体抗病毒信号蛋白(mitochondrial antiviral signaling protein,MAVS),启动下游信号级联反应,合成一系列的细胞因子,如I型干扰素和炎性细胞因子等,使宿主建立抗病毒天然免疫状态。目前,对DTMUV的致病性还不清楚,DTMUV感染后诱导的宿主天然免疫反应以及RLR在病毒感染过程中的作用更是缺乏了解。本研究以樱桃谷雏鸭为实验动物,通过病理组织学分析、荧光定量PCR等方法,首先观察DTMUV对不同日龄樱桃谷鸭的致病性差异,以及感染后诱导的天然免疫反应,然后使用过表达、RNA干扰和双荧光素酶报告基因检测等方法对RIG-I、MDA5和MAVS在病毒感染过程中的作用进行分析,初步探讨了DTMUV对RLR信号通路的抑制作用。研究内容主要分为以下四个方面:1.DTMUV对不同日龄樱桃谷鸭的致病性研究1、3和7周龄的樱桃谷鸭通过腿部肌肉注射途径感染DTMUV,然后观察感染鸭的临床症状、病理组织学变化、组织含毒量以及血清中和抗体等变化规律。结果发现,1周龄雏鸭在感染后第2天出现明显的以神经紊乱为主的临床症状,在感染后第5-7天,部分感染鸭死亡。3周龄感染鸭表现类似但程度较轻的临床症状,且未见死亡。而7周龄感染鸭仅出现一过性的食欲减退。实验鸭主要组织的病变程度随着感染日龄的增大而逐渐减轻。1周龄感染鸭出现明显的心内膜出血、脾肿大,回肠淋巴组织集中处黏膜肿胀,肝、肾肿胀且有出血,脑膜充血。3周龄感染鸭的病变程度较轻微,7周龄鸭则未见明显的眼观病变。但是,病理组织学分析显示,三组感染鸭均呈现典型的病毒性脑炎病变。组织中病毒含量检测发现,感染后第1天,DTMUV即能在1周龄感染鸭的大脑中检测到,感染后第3-5天,1周龄鸭的主要组织含毒量均显著高于另外两组感染鸭对应组织的含毒量(P0.05)。感染鸭血清中IFN-γ、IL-2及中和抗体的含量呈相似性变化,但1周龄鸭与另外两组鸭存在显著性差异(P0.05)。更重要的是,尽管各组实验鸭的血清中和抗体在DTMUV感染后第7-19天无显著差异,但是1周龄鸭的病毒清除速度却显著慢于3、7周龄鸭,表明与鸭日龄相关的自身免疫系统即天然免疫在感染早期清除病毒的过程中发挥关键作用。总的来说,本部分研究证实DTMUV对不同日龄鸭的致病性不同,日龄越小致病性越强,与日龄相关的免疫反应能力对DTMUV的致病性具有重要影响。2.DTMUV感染雏鸭和鸭胚成纤维细胞(DEFs)后诱导的天然免疫反应为确定DTMUV感染后诱导的天然免疫反应,我们通过相对荧光定量PCR检测了病毒感染1周龄雏鸭脾和脑组织中天然免疫相关基因的变化。结果显示,在DTMUV感染的早期阶段,病毒能显著上调RIG-I、MDA5和TLR3的表达(P0.05),说明这些PRRs参与DTMUV感染后诱导的天然免疫反应。同时,炎性细胞因子(IL-1β、L-2、IL-6和IL-8)和抗病毒蛋白(Mx、OAS等)也大量表达,其中IL-6表达量的增加最为显著。在感染鸭的脾和脑中,I型和II型IFNs的变化不同。在脾中,两种类型IFNs均呈现不同程度的上调,但是在脑中,二者变化趋势差异较大。除此之外,我们还检测了DTMUV体外感染DEFs后RIG-I、MDA5、I型IFNs和部分促炎细胞因子的表达变化,结果与体内实验类似,RIG-I和MDA5被病毒激活而过量表达,导致IFN-α和IFN-β、抗病毒蛋白OAS以及IL-6、IL-8等的大量表达。上述研究结果说明,DTMUV感染雏鸭和DEFs后其抗病毒天然免疫反应被迅速激活,但仍不足以抵抗病毒短时间内快速、大量的增殖,同时,促炎细胞因子如IL-6等的大量产生亦能对宿主自身造成一定的免疫损伤,从而加重病情。3.RIG-I和MDA5参与宿主的抗DTMUV感染为了进一步明确RIG-I和MDA5在抗DTMUV感染过程中的作用,使用过表达和双荧光素酶报告基因检测等技术分析了病毒感染DEFs后二者介导的IFN-β的产生情况。首先通过前期的实验,我们发现DTMUV能诱导RIG-I、MDA5、MAVS和IFN-β在m RNA水平上显著上调。进一步地,先在DEFs上分别过表达RIG-I和MDA5的全长及其效应结构域,再感染DTMUV,发现两个受体及其效应结构域均能抑制DTMUV增殖。继而,我们克隆并表达了鸭MAVS基因(du MAVS),通过过表达和RNA干扰证实,du MAVS在DTMUV感染过程中是必须的。此外,DTMUV感染后能进一步刺激RIG-I和MDA5诱导的转录因子NF-κB和IRF-7的产量,说明过表达的RIG-I和MDA5识别DTMUV核酸后能通过NF-κB和IRF-7信号通路激活下游IFN-β。本部分研究初步确定RIG-I/MDA5-MAVS-IRF-7/NF-κB信号通路介导IFN-β参与抗DTMUV的天然免疫反应。4.DTMUV的非结构蛋白NS1抑制RIG-I和MDA5介导的信号通路前期发现预先感染了DTMUV的细胞,再次经RIG-I或MDA5刺激后,其诱导的IFN-β表达量显著低于对照组细胞,提示DTMUV对RIG-I和MDA5介导的IFN-β的产生存在一定的抑制作用。为了进一步明确DTMUV通过何种方式拮抗宿主IFN-β的产生,我们分别克隆并构建了病毒7个非结构蛋白NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5的真核表达质粒,实验发现,过表达有NS1蛋白的细胞中IFN-β含量较对照组显著下降,表明DTMUV的NS1蛋白在拮抗RIG-I和MDA5的信号通路中起主要作用。进一步地,将NS1和du MAVS的真核表达质粒共转染DEFs,转染后24 h,通过激光共聚焦观察发现,DTMUV的NS1和du MAVS在细胞中存在共定位现象,初步确定DTMUV的NS1蛋白可能是通过与du MAVS的相互作用抑制了RLR信号通路。但是,此部分结果还需要通过免疫共沉淀等试验进行进一步的验证。
[Abstract]:Duck Tembusu virus (duck Tembusu, virus, DTMUV) is an enveloped, single stranded RNA virus belonging to the Flaviviridae flavivirus.DTMUV in 2010 first in Fujian, isolated from diseased duck field in Zhejiang and other places, mainly caused by the laying ducks egg drop, duck growth retardation and neurological symptoms. The disease spread rapidly, causing huge economic losses to our country. One of the most natural host immune responses in the early infection of pathogenic microorganisms play an important role in pattern recognition receptors (pattern recognition, receptors, PRRs) is the key of the host innate immune system components, conservative constituents of.RIG-I like receptor recognition the main pathogenic microorganisms (RIG-I like receptor, RLR) is a kind of PRRs was mainly distributed in the cytoplasm of non immune cells, including retinoic acid inducible gene I (retinoic acid induced gene-I, RIG-I), melanoma differentiation associated gene (M Elanoma differentiation associated gene 5, MDA5) and the genetic and physiological experiment gene 2 (Laboratory of genetics and physiology-2, LGP2), mainly through the joint identification of RNA virus, molecular mitochondrial antiviral signaling protein (mitochondrial antiviral signaling protein, MAVS), and activate the downstream signaling cascade of cytokine synthesis a series, such as type I interferon and inflammatory cytokines, the host innate immune antiviral state established. At present, the pathogenicity of DTMUV is unclear, DTMUV induced after infection of host innate immune reaction and RLR virus infection in the process is the lack of understanding. In this study, Yingtao Gu ducklings were used as the experimental animal, by histopathological analysis, fluorescence quantitative PCR methods, the first observation of pathogenicity of DTMUV to different ducks, as well as natural immunity after infection induced reaction, however After over expression, RNA interference and dual luciferase reporter gene assay method to analyze the role of MDA5 RIG-I, and MAVS in virus infection, investigate the inhibitory effects of DTMUV on RLR signal pathway. The main research contents are divided into the following four aspects: 1.DTMUV pathogenicity to ducks of different 1,3 and 7 week old Cherry Valley Duck through the legs intramuscular injection of DTMUV infection, clinical symptoms, and then observe the pathological changes of tissues of infected duck, virus content and serum neutralizing antibody were obtained. The results showed that 1 week old ducklings in infection occurred second days after clinical symptoms of neurological disorders mainly in the obvious. 5-7 days after infection, some infected duck deaths.3 weeks old infected duck were similar to clinical symptoms but to a lesser extent, and no death. At the age of 7 weeks and only infected ducks had transient experimental anorexia. The severity of the main organization with the increase of duck infection age decreased.1 week old infected duck appeared endocardial hemorrhage, splenomegaly, ileal lymphoid tissue concentration at the mucosal swelling, liver, kidney swelling and bleeding, the severity of congestion in.3 week old ducks infected meninges mild, 7 week old duck is not clear eye view lesions. However, histopathological analysis showed that three groups of infected ducks showed typical viral disease. Detection of virus content in tissues, first days after infection, DTMUV can be detected in the 1 week old duck infection in the brain, the 3-5 day after infection, the main organization of 1 week old ducks the virus content was significantly higher than that of the other two groups the amount of drug containing infected duck tissues (P0.05). The infection of duck serum levels of IFN-, IL-2 content and neutralization antibody showed a similar change, but there is a significant difference between the 1 week old duck and duck in the other two groups (P0.05). The more important Is that although serum neutralizing antibody in experimental duck DTMUV infection after 7-19 days of no significant difference, but the virus clearance rate of 1 week old ducks were significantly slower than the 3,7 week old duck, duck and indicate the age's own immune system that innate immunity plays a key role in the process of clearing the virus in the early stage of infection in general, this part of the study confirmed that the DTMUV of different pathogenicity of different day old duck, the smaller age of pathogenic stronger pathogenic immune response associated with the age of DTMUV has an important influence on.2.DTMUV Infected Ducklings and duck embryo fibroblast (DEFs) innate immune response to natural immune response after in order to determine the DTMUV induced after infection induced, we examined the change of virus infection natural immunity 1 week old ducklings of spleen and brain related genes by relative quantitative PCR. The results showed that in the early stage of DTMUV infection, virus Could increase the expression of MDA5 and RIG-I, TLR3 (P0.05), to explain the natural immune response induced by the PRRs in DTMUV after infection. At the same time, inflammatory cytokines (L-2, IL-6 and IL-1 beta, IL-8) and antiviral proteins (Mx, OAS) is widely expressed, the expression of IL-6 increased the most significant. In infected duck spleen and brain, the change of I type and II type of IFNs. In the spleen, two types of IFNs showed different degrees of increase, but in the brain, the change trend of the two big difference. In addition, we also examined DTMUV in vitro after infection with DEFs RIG-I, MDA5 I, IFNs and proinflammatory cytokines part expression results similar to the in vivo experiment, RIG-I and MDA5 are activated by viruses and excessive expression, leading to IFN- alpha and IFN- beta, antiviral protein OAS and IL-6, a large number of IL-8. The results showed that, DTMUV Infected Ducklings and DEFs after its antiviral innate immunity Immune response is activated rapidly, but still not enough to resist the virus quickly in a short time, a large number of proliferation, at the same time, a large number of proinflammatory cytokines such as IL-6 can also cause certain immune injury to host itself, thus aggravating the condition of.3.RIG-I and MDA5 in the host anti DTMUV infection in order to further clarify the role of RIG-I and MDA5 in the anti DTMUV infection, using overexpression and dual luciferase reporter gene assay technique to analyze the virus infection DEFs two mediated IFN- beta. Firstly, through the experiment, we found that DTMUV can induce RIG-I, MDA5, MAVS and IFN- increased significantly in M beta RNA level further. First, in DEFs respectively and the effect of overexpression of full-length domains RIG-I and MDA5, and then infected with DTMUV, found two receptor and effector domain could inhibit the proliferation of DTMUV. Then, we cloned and expressed the duck MAV S gene (DU MAVS), the expression of RNA and Du confirmed that the interference of MAVS is necessary in the process of DTMUV infection. In addition, DTMUV infection can stimulate RIG-I and MDA5 induced NF- transcription factor kappa B and IRF-7 yield, that over expression of RIG-I and MDA5 to identify DTMUV nucleic acid can activate the downstream IFN- Beta Kappa B via NF- and IRF-7 signaling pathways. This part of the study identified RIG-I/MDA5-MAVS-IRF-7/NF- signaling pathway B signaling pathway mediated by IFN- is involved in the innate immune response against.4.DTMUV DTMUV non structural protein NS1 inhibits RIG-I and MDA5 mediated early discovery of pre infected DTMUV cells, again after RIG-I or MDA5 stimulation. The induced expression of IFN- was significantly lower than the control group cells, suggesting that DTMUV of RIG-I and MDA5 mediated IFN- beta had certain inhibitory effect. In order to further clarify the means by which DTMUV host IFN- beta antagonists We have cloned and constructed 7 virus non structural protein NS1, NS2A, NS2B, NS3, NS4A, eukaryotic expression plasmid, NS4B and NS5 found that over expression of NS1 protein in cells of IFN- beta content significantly decreased compared to the control group, DTMUV showed that NS1 protein play a major role in the signaling pathway antagonism of RIG-I and MDA5. Furthermore, NS1 and Du MAVS eukaryotic expression plasmids were transfected into DEFs, 24 h after transfection by laser confocal observation, DTMUV NS1 and Du MAVS were colocalized in cells, initially identified DTMUV NS1 protein may be through interaction with Du MAVS the inhibition of RLR signaling pathway. However, the results of this part are validated by CO immunoprecipitation experiments.

【学位授予单位】：山东农业大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S852.65

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